Methods of producing enriched populations of tumor reactive T cells from peripheral blood

ABSTRACT

Methods of obtaining a cell population enriched for tumor-reactive T cells, the method comprising: (a) obtaining a bulk population of peripheral blood mononuclear cells (PBMCs) from a sample of peripheral blood; (b) specifically selecting CD8 + T cells that also express PD-1 and/or TIM-3 from the bulk population; and (c) separating the cells selected in (b) from unselected cells to obtain a cell population enriched for tumor-reactive T cells are disclosed. Related methods of administering a cell population enriched for tumor-reactive T cells to a mammal, methods of obtaining a pharmaceutical composition comprising a cell population enriched for tumor-reactive T cells, and isolated or purified cell populations are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a U.S. National Phase of International PatentApplication No. PCT/US2013/038813, filed Apr. 30, 2013, which claims thebenefit of U.S. Provisional Patent Application No. 61/771,251, filedMar. 1, 2013, each of which is incorporated by reference in its entiretyherein.

BACKGROUND OF THE INVENTION

Adoptive cell therapy (ACT) using tumor reactive T cells can producepositive clinical responses in some cancer patients. Nevertheless,several obstacles to the successful use of ACT for the treatment ofcancer and other diseases remain. For example, T cells isolated fromperipheral blood may not exhibit sufficient tumor-specific reactivity.Accordingly, there is a need for improved methods of obtaining apopulation of tumor reactive T cells from peripheral blood.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides a method of obtaining a cellpopulation enriched for tumor-reactive T cells, the method comprising:(a) obtaining a bulk population of peripheral blood mononuclear cells(PBMCs) from a sample of peripheral blood; (b) specifically selectingCD8⁺ T cells that also express PD-1 and/or TIM-3 from the bulkpopulation; and (c) separating the cells selected in (b) from unselectedcells to obtain a cell population enriched for tumor-reactive T cells.

Another embodiment of the invention provides a method of administering acell population enriched for tumor-reactive T cells to a mammal, themethod comprising: (a) obtaining a bulk population of PBMCs from asample of peripheral blood; (b) specifically selecting CD8⁺ T cells thatalso express PD-1 and/or TIM-3 from the bulk population; (c) separatingthe cells selected in (b) from unselected cells to obtain a cellpopulation enriched for tumor-reactive T cells; and (d) administeringthe cell population enriched for tumor-reactive T cells to the mammal.

Still another embodiment of the invention provides a method of obtaininga pharmaceutical composition comprising a cell population enriched fortumor-reactive T cells, the method comprising: (a) obtaining a bulkpopulation of PBMCs from a sample of peripheral blood; (b) specificallyselecting CD8⁺ T cells that also express PD-1 and/or TIM-3 from the bulkpopulation; (c) separating the cells selected in (b) from unselectedcells to obtain a cell population enriched for tumor-reactive T cells;and (d) combining the cell population enriched for tumor-reactive Tcells with a pharmaceutically acceptable carrier to obtain apharmaceutical composition comprising a cell population enriched fortumor-reactive T cells.

Additional embodiments of the invention provide related populations ofcells and methods of treating or preventing cancer.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A-1B are graphs showing interferon (IFN)-gamma secretion (pg/ml)(black bars) and percentage of CD8⁺ cells expressing 4-1BB (grey bars)by cells that were isolated from the peripheral blood of melanomapatient 1913 (A) or melanoma patient 3713 (B) and which were sorted forexpression of CD8, PD-1, or TIM-3 or lack of expression of PD-1 or TIM-3by fluorescence-activated cell sorting (FACS) and expanded in vitro,upon co-culture against the autologous tumor cell line.

FIG. 1C is a graph showing IFN-gamma secretion (pg/ml) (black bars) andpercentage of CD8⁺ cells expressing 4-1BB (grey bars) by cells that wereisolated from the peripheral blood of melanoma patient 3289 and whichwere sorted for expression of CD8, PD-1, LAG-3, or TIM-3 or lack ofexpression of PD-1 or TIM-3 by FACS, upon co-culture against theautologous tumor.

FIGS. 2A-2C are graphs showing percent specific lysis of targetautologous tumor cell line TC1913 (A), allogeneic (Allo.) tumor cellline TC3289 (B), or HLA-A0201-matched tumor cell line TC2448 (C) byeffector cells isolated from the peripheral blood of patient 1913,sorted for expression as follows: CD8⁺ (open circles), PD-1⁺ (blackcircles), PD-1⁻ (grey circles), TIM-3⁺ (black diamonds), or TIM-3⁻ (greydiamonds), and expanded in vitro.

FIGS. 2D-2F are graphs showing percent specific lysis of targetautologous tumor cell line TC3289 (D), allogeneic tumor cell line TC1913(E), or allogeneic tumor cell line TC624 (F) by effector cells isolatedfrom the peripheral blood of patient 3289 (D-F) and sorted forexpression as follows: CD8⁺ (open circles), PD-1⁺ (black circles), PD-1⁻(grey circles), TIM-3⁺ (black diamonds), or TIM-3⁻ (grey diamonds).Target cell lysis after in vitro expansion is shown.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that selecting CD8⁺ cells that also expressprogrammed cell death protein 1 (PD-1; CD279) and/or T-cellimmunoglobulin and mucin domain 3 (TIM-3) biomarkers enriches fortumor-reactive T cells present in peripheral blood. Selecting the CD8⁺cells that also express PD-1 and/or TIM-3 advantageously enriches forgreater numbers of tumor-reactive T cells as compared to CD8⁺ cells thatdo not express these markers.

In this regard, an embodiment of the invention provides a method ofobtaining a cell population enriched for tumor-reactive T cells, themethod comprising: (a) obtaining a bulk population of peripheral bloodmononuclear cells (PBMCs) from a sample of peripheral blood; (b)specifically selecting CD8⁺ T cells that also express PD-1 and/or TIM-3from the bulk population; and (c) separating the cells selected in (b)from unselected cells to obtain a cell population enriched fortumor-reactive T cells. The inventive methods advantageously make itpossible to shorten the time of in vitro culture and to select fortumor-reactive T cells without having to screen for autologous tumorrecognition.

The method may comprise obtaining a bulk population of PBMCs from asample of peripheral blood by any suitable method known in the art.Suitable methods of obtaining a bulk population of PBMCs may include,but are not limited to, a blood draw and/or a leukapheresis. The bulkpopulation of PBMCs obtained from a tumor sample may comprise T cells,including tumor-reactive T cells.

The peripheral blood may be obtained from any mammal. Unless statedotherwise, as used herein, the term “mammal” refers to any mammalincluding, but not limited to, mammals of the order Logomorpha, such asrabbits; the order Carnivora, including Felines (cats) and Canines(dogs); the order Artiodactyla, including Bovines (cows) and Swines(pigs); or of the order Perssodactyla, including Equines (horses). It ispreferred that the mammals are non-human primates, e.g., of the orderPrimates, Ceboids, or Simoids (monkeys) or of the order Anthropoids(humans and apes). In some embodiments, the mammal may be a mammal ofthe order Rodentia, such as mice and hamsters. Preferably, the mammal isa non-human primate or a human. An especially preferred mammal is thehuman.

The method may comprise specifically selecting CD8⁺ T cells that alsoexpress PD-1 and/or TIM-3 from the bulk population. In a preferredembodiment, the method comprises selecting cells that also express CD3.The method may comprise specifically selecting the cells in any suitablemanner. Preferably, the selecting is carried out using flow cytometry.The flow cytometry may be carried out using any suitable method known inthe art. The flow cytometry may employ any suitable antibodies andstains. For example, the specific selection of CD3, CD8, TIM-3, or PD-1may be carried out using anti-CD3, anti-CD8, anti-TIM-3, or anti-PD-1antibodies, respectively. Preferably, the antibody is chosen such thatit specifically recognizes and binds to the particular biomarker beingselected. The antibody or antibodies may be conjugated to a bead (e.g.,a magnetic bead) or to a fluorochrome. Preferably, the flow cytometry isfluorescence-activated cell sorting (FACS).

In an embodiment of the invention, specifically selecting may comprisespecifically selecting CD8⁺ T cells that are positive for expression ofany one of TIM-3, PD-1, or both TIM-3 and PD-1. In this regard,specifically selecting may comprise specifically selecting T cells thatare single positive for expression of TIM-3 or PD-1 or double positivefor simultaneous co-expression of TIM-3 and PD-1. In an embodiment ofthe invention, the method comprises specifically selecting CD8⁺ T cellsthat express TIM-3 from the bulk population. In still another embodimentof the invention, the method comprises specifically selecting CD8⁺ Tcells that express PD-1 from the bulk population. Still anotherembodiment of the invention comprises specifically selecting CD8⁺ Tcells that are (i) TIM-3⁺/PD-1⁺, (ii) TIM-3⁻/PD-1⁺, or (iii)TIM-3⁺/PD-1⁻ from the bulk population. In another embodiment of theinvention, any of the methods described herein may further compriseselecting cells that also express CD3⁺.

In an embodiment of the invention, specifically selecting may comprisespecifically selecting combinations of CD8⁺ cells expressing any of themarkers described herein. In this regard, the method may produce a cellpopulation that is enriched for tumor-reactive cells that comprises amixture of cells expressing any two of the biomarkers described herein.In an embodiment of the invention, specifically selecting comprisesspecifically selecting a combination of PD-1⁺ cells and TIM-3⁺ cells. Inanother embodiment of the invention, any of the methods described hereinmay further comprise selecting cells that also express CD8⁺ and/or CD3⁺.

The method may comprise separating the selected cells from unselectedcells to obtain a cell population enriched for tumor-reactive T cells.In this regard, the selected cells may be physically separated from theunselected cells. The selected cells may be separated from unselectedcells by any suitable method such as, for example, sorting. Separatingthe selected cells from the unselected cells preferably produces a cellpopulation that is enriched for tumor-reactive T cells.

The cell populations obtained by the inventive methods areadvantageously enriched for tumor-reactive T cells. In this regard, thecell populations obtained by the inventive methods may comprise a higherproportion of tumor reactive T cells as compared to cell populationsthat have not been obtained by sorting for expression of TIM-3 and/orPD-1.

In an embodiment of the invention, the method comprises obtaining thecell population enriched for tumor-reactive T cells without screeningfor autologous tumor recognition. In this regard, the inventive methodsadvantageously provide a cell population that is enriched for cells thathave tumor reactivity without having to screen the cells for autologoustumor recognition.

In an embodiment of the invention, the method does not comprisenon-specifically stimulating the bulk population of T cells prior tospecifically selecting the cells. In this regard, the inventive methodsadvantageously provide a cell population that is enriched for tumorreactive T cells without stimulating the bulk population of T cellsnonspecifically (e.g., with anti-4-1BB antibodies, anti-CD3 antibodies,and/or anti-CD28 antibodies).

In an embodiment of the invention, the method further comprisesexpanding the numbers of T cells in the enriched cell populationobtained by the inventive methods in vitro. The numbers of T cells maybe increased at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold),more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-,80-, or 90-fold), more preferably at least about 100-fold, morepreferably at least about 1,000 fold, or most preferably at least about100,000-fold. The numbers of T cells may be expanded using any suitablemethod known in the art. Exemplary methods of expanding the numbers ofcells are described in U.S. Pat. No. 8,034,334 and U.S. PatentApplication Publication No. 2012/0244133, each of which is incorporatedherein by reference.

In an embodiment of the invention, the method further comprisesculturing the enriched cell population obtained by the inventive methodsin the presence of any one or more of TWS119, interleukin (IL-21),IL-12, IL-15, IL-7, transforming growth factor (TGF) beta, and AKTinhibitor (AKTi). Without being bound to a particular theory, it isbelieved that culturing the enriched cell population in the presence ofTWS119, IL-21, and/or IL-12 may, advantageously, enhance the anti-tumorreactivity of the enriched cell population by preventing or retardingthe differentiation of the enriched cell population.

In an embodiment of the invention, the method further comprisestransducing or transfecting the cells of the enriched populationobtained by any of the inventive methods described herein with anucleotide sequence encoding any one or more of IL-12, IL-7, IL-15,IL-2, IL-21, mir155, and anti-PD-1 siRNA.

In an embodiment of the invention, the method further comprisesstimulating the enriched cell population obtained by the inventivemethods with a cancer antigen and/or with autologous tumor cells.Stimulating the enriched cell population with a cancer antigen and/orwith autologous tumor cells may be carried out by any suitable method.For example, stimulating the enriched cell population may be carried outby physically contacting the enriched cell population with a cancerantigen and/or with autologous tumor cells. Without being bound to aparticular theory, it is believed that stimulating the enriched cellpopulation with a cancer antigen and/or with autologous tumor cells may,advantageously, enhance the anti-tumor reactivity of the enriched cellpopulation.

The term “cancer antigen” as used herein refers to any molecule (e.g.,protein, peptide, lipid, carbohydrate, etc.) solely or predominantlyexpressed or over-expressed by a tumor cell or cancer cell, such thatthe antigen is associated with the tumor or cancer. The cancer antigencan additionally be expressed by normal, non-tumor, or non-cancerouscells. However, in such cases, the expression of the cancer antigen bynormal, non-tumor, or non-cancerous cells is not as robust as theexpression by tumor or cancer cells. In this regard, the tumor or cancercells can over-express the antigen or express the antigen at asignificantly higher level, as compared to the expression of the antigenby normal, non-tumor, or non-cancerous cells. Also, the cancer antigencan additionally be expressed by cells of a different state ofdevelopment or maturation. For instance, the cancer antigen can beadditionally expressed by cells of the embryonic or fetal stage, whichcells are not normally found in an adult host. Alternatively, the cancerantigen can be additionally expressed by stem cells or precursor cells,which cells are not normally found in an adult host.

The cancer antigen can be an antigen expressed by any cell of any canceror tumor, including the cancers and tumors described herein. The cancerantigen may be a cancer antigen of only one type of cancer or tumor,such that the cancer antigen is associated with or characteristic ofonly one type of cancer or tumor. Alternatively, the cancer antigen maybe a cancer antigen (e.g., may be characteristic) of more than one typeof cancer or tumor. For example, the cancer antigen may be expressed byboth breast and prostate cancer cells and not expressed at all bynormal, non-tumor, or non-cancer cells. Exemplary cancer antigens mayinclude any one or more of gp100, MART-1, MAGE-A1, MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, NY-ESO-1, vascular endothelial growth factorreceptor-2 (VEGFR-2), HER-2, mesothelin, and epidermal growth factorreceptor variant III (EGFR III).

The inventive methods advantageously produce cell populations enrichedfor tumor-reactive T cells. The T cells may be tumor-reactive such thatthey specifically recognize, lyse, and/or and kill tumor cells. In thisregard, an embodiment of the invention provides an isolated or purifiedcell population enriched for tumor-reactive T cells obtained by any ofthe inventive methods described herein. In an embodiment, the isolatedor purified cell population comprises (a) CD8⁺/TIM-3⁺/PD-1⁺ T cells, (b)CD8⁺/TIM-3⁻/PD-1⁺ T cells, and (c) CD8⁺/TIM-3⁺/PD-1⁻ T cells, whereinthe cell population is enriched for tumor-reactive T cells. In anotherembodiment of the invention, the isolated or purified cell populationcomprises (a) CD8⁺/TIM-3⁺/PD-1⁺ T cells, (b) CD8⁺/TIM-3⁻/PD-1⁺ T cells,or (c) CD8⁺/TIM-3⁺/PD-1⁻ T cells. In another embodiment of theinvention, any of the cell populations described herein may also beCD3⁺.

In an embodiment of the invention, the isolated or purified cellpopulation comprises a mixture of cells expressing any of the biomarkersdescribed herein. For example, the isolated or purified cell populationmay comprise a combination of PD-1⁺ cells and TIM-3⁺ cells. In anotherembodiment of the invention, any of the cell populations describedherein may also be CD8⁺ and/or CD3⁺.

The term “isolated” as used herein means having been removed from itsnatural environment. The term “purified” as used herein means havingbeen increased in purity, wherein “purity” is a relative term, and notto be necessarily construed as absolute purity. For example, the puritycan be at least about 50%, can be greater than 60%, 70% or 80%, 90% orcan be 100%.

Another embodiment of the invention provides a method of administering acell population enriched for tumor-reactive T cells to a mammal, themethod comprising: (a) obtaining a bulk population of PBMCs from asample of peripheral blood; (b) specifically selecting CD8⁺ T cells thatalso express PD-1 and/or TIM-3 from the bulk population; (c) separatingthe cells selected in (b) from unselected cells to obtain a cellpopulation enriched for tumor-reactive T cells; and (d) administeringthe cell population enriched for tumor-reactive T cells to the mammal.Obtaining a bulk population of PBMCs from a sample of peripheral blood,specifically selecting CD8⁺ T cells that also express PD-1 and/or TIM-3from the bulk population, and separating the selected cells fromunselected cells to obtain a cell population enriched for tumor-reactiveT cells may be carried out as described herein with respect to otheraspects of the invention.

The method may further comprise administering the cell populationenriched for tumor-reactive T cells to the mammal. The cell populationenriched for tumor-reactive T cells may be administered in any suitablemanner. Preferably, the cell population enriched for tumor-reactive Tcells is administered by injection, e.g., intravenously.

The inventive cell population enriched for tumor-reactive T cells can beincluded in a composition, such as a pharmaceutical composition. In thisregard, the invention provides a pharmaceutical composition comprisingany of the cell populations described herein and a pharmaceuticallyacceptable carrier.

Another embodiment of the invention provides a method of obtaining apharmaceutical composition comprising a cell population enriched fortumor-reactive T cells, the method comprising: (a) obtaining a bulkpopulation of PBMCs from a sample of peripheral blood; (b) specificallyselecting CD8⁺ T cells that also express PD-1 and/or TIM-3 from the bulkpopulation; (c) separating the cells selected in (b) from unselectedcells to obtain a cell population enriched for tumor-reactive T cells;and (d) combining the cell population enriched for tumor-reactive Tcells with a pharmaceutically acceptable carrier to obtain apharmaceutical composition comprising a cell population enriched fortumor-reactive T cells. Obtaining a bulk population of PBMCs from asample of peripheral blood, specifically selecting CD8⁺ T cells thatalso express PD-1 and/or TIM-3 from the bulk population, and separatingthe selected cells from unselected cells to obtain a cell populationenriched for tumor-reactive T cells may be carried out as describedherein with respect to other aspects of the invention.

The method may comprise combining the cell population enriched fortumor-reactive T cells with a pharmaceutically acceptable carrier toobtain a pharmaceutical composition comprising a cell populationenriched for tumor-reactive T cells. Preferably, the carrier is apharmaceutically acceptable carrier. With respect to pharmaceuticalcompositions, the carrier can be any of those conventionally used forthe administration of cells. Such pharmaceutically acceptable carriersare well-known to those skilled in the art and are readily available tothe public. It is preferred that the pharmaceutically acceptable carrierbe one which has no detrimental side effects or toxicity under theconditions of use. A suitable pharmaceutically acceptable carrier forthe cells for injection may include any isotonic carrier such as, forexample, normal saline (about 0.90% w/v of NaCl in water, about 300mOsm/L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOLR electrolyte solution (Abbott, Chicago, Ill.), PLASMA-LYTE A (Baxter,Deerfield, Ill.), about 5% dextrose in water, or Ringer's lactate. In anembodiment, the pharmaceutically acceptable carrier is supplemented withhuman serum albumen.

For purposes of the invention, the dose, e.g., number of cells in theinventive cell population enriched for tumor-reactive T cells,administered should be sufficient to effect, e.g., a therapeutic orprophylactic response, in the mammal over a reasonable time frame. Forexample, the number of cells should be sufficient to bind to a cancerantigen, or detect, treat or prevent cancer in a period of from about 2hours or longer, e.g., 12 to 24 or more hours, from the time ofadministration. In certain embodiments, the time period could be evenlonger. The number of cells will be determined by, e.g., the efficacy ofthe particular cells and the condition of the mammal (e.g., human), aswell as the body weight of the mammal (e.g., human) to be treated.

Many assays for determining an administered number of cells from theinventive cell population enriched for tumor-reactive T cells are knownin the art. For purposes of the invention, an assay, which comprisescomparing the extent to which target cells are lysed or one or morecytokines such as, e.g., IFN-γ and IL-2 are secreted upon administrationof a given number of such cells to a mammal, among a set of mammals ofwhich is each given a different number of the cells, could be used todetermine a starting number to be administered to a mammal. The extentto which target cells are lysed, or cytokines such as, e.g., IFN-γ andIL-2 are secreted, upon administration of a certain number of cells, canbe assayed by methods known in the art. Secretion of cytokines such as,e.g., IL-2, may also provide an indication of the quality (e.g.,phenotype and/or effectiveness) of a cell preparation.

The number of the cells from the inventive cell population enriched fortumor-reactive T cells also will be determined by the existence, natureand extent of any adverse side effects that might accompany theadministration of a particular cell population. Typically, the attendingphysician will decide the number of the cells with which to treat eachindividual patient, taking into consideration a variety of factors, suchas age, body weight, general health, diet, sex, route of administration,and the severity of the condition being treated. By way of example andnot intending to limit the invention, the number of cells can be about10×10⁶ to about 10×10¹¹ cells per infusion, about 10×10⁹ cells to about10×10¹¹ cells per infusion, or 10×10⁷ to about 10×10⁹ cells perinfusion. The cell populations obtained by the inventive methods may,advantageously, make it possible to effectively treat or prevent cancer.

It is contemplated that the cell populations obtained by the inventivemethods can be used in methods of treating or preventing cancer. In thisregard, the invention provides a method of treating or preventing cancerin a mammal, comprising administering to the mammal the pharmaceuticalcompositions or cell populations obtained by any of the inventivemethods described herein in an amount effective to treat or preventcancer in the mammal. Another embodiment of the invention provides amethod of treating or preventing cancer in a mammal, comprisingadministering a cell population enriched for tumor-reactive T cells to amammal by any of the inventive methods described herein in an amounteffective to treat or prevent cancer in the mammal.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount or any level of treatment or preventionof cancer in a mammal. Furthermore, the treatment or prevention providedby the inventive method can include treatment or prevention of one ormore conditions or symptoms of the disease, e.g., cancer, being treatedor prevented. Also, for purposes herein, “prevention” can encompassdelaying the onset of the disease, or a symptom or condition thereof.

For purposes of the inventive methods, wherein populations of cells areadministered, the cells can be cells that are allogeneic or autologousto the mammal. Preferably, the cells are autologous to the mammal.

An embodiment of the invention further comprises lymphodepleting themammal prior to administering any of the enriched cell populationsobtained by any of the inventive methods described herein. Examples oflymphodepletion include, but may not be limited to, nonmyeloablativelymphodepleting chemotherapy, myeloablative lymphodepletingchemotherapy, total body irradiation, etc.

With respect to the inventive methods, the cancer can be any cancer,including any of sarcomas (e.g., synovial sarcoma, osteogenic sarcoma,leiomyosarcoma uteri, and alveolar rhabdomyosarcoma), lymphomas (e.g.,Hodgkin lymphoma and non-Hodgkin lymphoma), hepatocellular carcinoma,glioma, head-neck cancer, acute lymphocytic cancer, acute myeloidleukemia, bone cancer, brain cancer, breast cancer, cancer of the anus,anal canal, or anorectum, cancer of the eye, cancer of the intrahepaticbile duct, cancer of the joints, cancer of the neck, gallbladder, orpleura, cancer of the nose, nasal cavity, or middle ear, cancer of theoral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronicmyeloid cancer, colon cancer (e.g., colon carcinoma), esophageal cancer,cervical cancer, gastrointestinal cancer (e.g., gastrointestinalcarcinoid tumor), hypopharynx cancer, larynx cancer, liver cancer, lungcancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynxcancer, ovarian cancer, pancreatic cancer, peritoneum, omentum, andmesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renalcancer, small intestine cancer, soft tissue cancer, stomach cancer,testicular cancer, thyroid cancer, ureter cancer, and urinary bladdercancer.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example demonstrates the in vitro autologous tumor recognition of Tcells isolated from the peripheral blood of melanoma patients accordingto expression of PD-1, TIM-3, or LAG-3 after expanding the numbers ofcells in vitro.

4-1BB up-regulation is an indicator of TCR stimulation. It has beenobserved that after the numbers of cells are expanded and in the absenceof TCR stimulation, 4-1BB expression is lost. It has also been observedthat after the numbers of cells are expanded and the cells areco-cultured with the autologous tumor cell line, T cells that hadpreviously lost 4-1BB expression and which are stimulated by the cellline will re-express 4-1BB. Accordingly, 4-1BB expression is measured 24hours after co-culture with autologous tumor as a marker of TCRstimulation against the autologous tumor cell line.

Cells obtained from the peripheral blood of each of three melanomapatients (1913, 3713, and 3289) by apheresis were rested over nightwithout cytokines and stained. The cells from patients 1913 and 3713were sorted into the following CD3⁺ populations using anti-CD3,anti-CD8, anti-PD-1, and TIM-3 antibodies: CD8⁺, CD8⁺/PD-1⁺,CD8⁺/TIM-3⁺, CD8⁺/PD-1⁻, or CD8⁺/TIM-3⁻ by fluorescence-activated cellsorting (FACS). The cells from patient 3289 were sorted into thefollowing CD3⁺ populations using anti-CD3, anti-CD8, anti-PD-1, TIM-3,and LAG-3 antibodies: CD8⁺, CD8⁺/PD-1⁺, CD8⁺/LAG3⁺, CD8⁺/TIM-3⁺,CD8⁺/PD-1⁻, CD8⁺/LAG3⁻, or CD8⁺/TIM-3⁻ by FACS. The numbers of cellswere expanded in vitro for 14 days. On day 14, cells were washed andco-cultured with the corresponding autologous tumor cell lines (1×10⁵effectors: 1×10⁵ target cells) and reactivity was assessed byquantifying IFN-gamma release and percentage of CD8⁺ cells expressing 41BB 24 hours after co-culture. The results are shown in FIGS. 1A-1C andTables 1-3. As shown in FIGS. 1A-1C and Tables 1-3, cells sortedaccording to expression of PD-1 or TIM-3 were enriched fortumor-reactive cells as compared to cells that were negative for PD-1 orTIM-3 expression, respectively.

TABLE 1 TC 624 CIITA TC 624 TC 1913 TC1913 FrTu#1913 FrTu#1913 Allo.CIITA T cells Aut. ⁺W6/32 Aut. ⁺W6/32 A0201 ⁺W6/32 T_(eff) PheresisPre-1913 CD8⁺ 13 342      7 (0.4) 1290 (1.4)  46 (0.5) 3942     33 (0.7)(0.3) (2.4) (3.4) PD-1⁺ 52 5693     56 (4.6) 2122 (14.1) 89 (1.8) 6890    15 (0.9) (1.3) (12.1)  (3.0) PD-1− 18 163     4 (0.5) 252 (1.0) 38(0.4) 2047      6 (0.3) (0.2) (1.6) (2.5) TIM-3⁺ 144 1303     15 (0.9)1150 (8.4)  138 (1.2)  1389     10 (0.4) (3.0) (15.8)  (1.3) TIM-3⁻ 0244     0 (0.6) 430 (1.1) 16 (0.3) 1754      1 (0.5) (0.1) (1.3) (2.1)TC 624 CIITA TC ⁺HLA- 2119 TC2448 TC1865 TC1379 TC2301 OKT3 DR A0201A0201 A0201 A11 Allo (0.1 μg/ml) T_(eff) Pheresis Pre-1913 CD8⁺ 2684    3099     935     1723     1301     328 67357     (2.1) (0.7) (1.9) (2.8)(3.6) (1.1) (88.4) PD-1⁺ 5610     933     413     174    43   240 53874    (2.3) (2.0) (1.1) (1.4) (0.8) (1.0) (86.1) PD-1− 1794     2457    439     528     158    202 41469     (1.7) (1.7) (2.0) (3.1) (2.9) (1.2)(89.1) TIM-3⁺ 1346     785     1124     113    50   46 69564     (1.6)(2.6) (5.4) (1.9) (2.9) (0.7) (92.2) TIM-3⁻ 1169     1427     1215    608     370     262 52940     (1.6) (0.7) (1.1) (3.1) (2.3) (1.1) (92.9)In vitro expanded effector populations isolated from peripheral blood ofPatient 1913 according to expression of the cell surface markersindicated were co-cultured against the autologous (Aut.) tumor cellsline (TC1913) and allogeneic (Allo.) tumor cells lines. Reactivity byIFN gamma (pg/ml) is shown. Values in parenthesis are the percentage ofCD3⁺ CD8⁺ cells that up-regulated CD137 (41BB) 24 hours (h) afterco-culture. Tumor cell lines (TC) 624 CIITA, 2119, 2448, and 1865 shareHLA A*0201 allele with TC1913, and TC 1379 shares A*11 with TC1913.TC2301 is an allogeneic control (mismatched for all HLA) used as anegative control. Values >300 pg/ml and greater than twice thebackground are considered positive and are shown underlined and in bold.

TABLE 2 TC624 CIITA TC624 TC624 HLA- CIITA CIITA A0201 ⁺W6/32 ⁺HLA-DRTC2119 T TC3713 TC3713 matched matched matched matched TC2119 TC1379TC3460 OKT3 cells Aut. ⁺W6/32 A*0201 A*0201 A*0201 A*0201 ⁺W6/32 Allo.Allo. (0.1 μ/ml) Pheresis CD3⁺ 0 (0) 25 (0.3) 0 (0)   149 (0.6) 0 (0.1)102 (0.2) 634 (1.2) 0 (0.2) 50 (0.7) 95 (0.1) 24946 CD8⁺    (71.9) PD-1⁺  0 (0.3) 713 (1.5)  0 (0.7)  68 (0.7) 0 (0.2)  53 (0.3) 186 (0.9) 0(0.1) 40 (1.2) 186 (0.6)  24044    (69.4) PD-1− 0 (0) 17 (0.1) 0 (0.1) 93 (0.4) 10 (0.2)  104 (0.4) 216 (0.9) 0 (0.1) 71 (0.7) 42 (0.1) 26901   (74.2) Tim-3⁺  31 (0.2) 32 (0)   5 (0.3) 247 (0.4) 8 (0.2) 198 (0.4)281 (0.4) 11 (0.2)  26 (0.4) 14 (0.2) 43218    (69.9) In vitro expandedeffector populations isolated from peripheral blood of patient 3713according to expression of the cell surface markers indicated wereco-cultured against the autologous tumor cells line (TC3713) andallogeneic tumor cells lines. Reactivity by IFN gamma (pg/ml) is shown.Values in parenthesis are the percentage of CD3⁺ CD8⁺ cells thatup-regulated CD137 (41BB) 24 h after co-culture. Tumor cell lines (TC)624 CIITA and 2119 share HLA A*0201 allele with TC3713. TC 1379 and TC3460 are allogeneic target cell lines (mismatched for all HLA) used as anegative control. Values >300 pg/ml and greater than twice thebackground are considered positive and are shown underlined and in bold.

TABLE 3 ⁺CD4⁺ ⁺TC3289 CD25− ⁺TC3289 ⁺TC3289 (IFN-γ ⁺TC1379 ⁺TC526 ⁺TC526Aut. normal OKT3 T cells Aut. ⁺W6/32 treatment Allo Allo ⁺W6/32 target(0.1 μ/ml) Pheresis CD3⁺ 120 (0.5)  69 (0.4) 44 (0.2) 128 (0.5) 37 (0.3)121 (0.6) 34 (0.2) 103 (0.8) 93232 (87.8) CD8⁺ PD-1⁺  47 (0.6) 1849(5.9)  1013 (1.8)  969 (3.5)  0 (0.5) 109 (0.8)  0 (0.1)  35 (2.3) 59617(90.9) PD-1− 157 (0.5)  85 (0.4) 58 (0.1) 125 (0.5) 66 (0.4) 164 (1.0)46 (0.2) 124 (1.0) 70009 (88.9) LAG-3⁺ 158 (0.8)  76 (0.8) 50 (0.3) 101(0.5) 29 (0.4)  71 (1.0) 30 (0.3) 124 (1.5) 84514 (78.4) LAG-3− 234(1.0) 102 (0.6) 57 (0.3) 132 (0.5) 56 (0.5)  95 (0.8) 43 (0.2) 144 (1.2)70004 (88.5) Tim-3⁺ 434 (2.2) 289 (2.4) 118 (0.4)  450 (1.6) 128 (1.1) 448 (1.4) 111 (0.4)  247 (2.2) 46674 (87.8) Tim-3− 196 (0.6) 103 (0.6)77 (0.2) 147 (0.4) 105 (0.6)  113 (0.8) 63 (0.2) 142 (1.2) 95104 (88.0)In vitro expanded effector populations isolated from the peripheralblood of Patient 3289 according to expression of the cell surfacemarkers indicated were co-cultured against the autologous tumor cellsline (3289) and allogeneic tumor cells lines. Reactivity by IFN gamma(pg/ml) is shown. Values in parenthesis are the percentage of CD3⁺ CD8⁺cells that up-regulated CD137 (41BB) 24 h after co-culture. Tumor celllines (TC) 1379 and TC526 are allogeneic cell lines (missmatched for allHLA) and autolgous CD4⁺ CD25⁻ cells isolated from peripheral blood usedas a negative control. Values >300 pg/ml and greater than twice thebackground are considered positive and are shown underlined and in bold.

EXAMPLE 2

This example demonstrates the in vitro autologous tumor recognition of Tcells isolated from the peripheral blood of melanoma patients and sortedaccording to expression of PD-1 or TIM-3 after expanding the numbers ofcells in vitro.

Cells were obtained from the peripheral blood of patient 1913 or patient3289 and were sorted according to expression of PD-1 or TIM-3 by FACS asdescribed in Example 1. The numbers of sorted cells were expanded for 14days in vitro. On day 15, target tumor cell lines (autologous andallogeneic) were labeled with ⁵¹Cr and co-cultured for 4 hours with thesorted populations of cells (effector cells) at the ratios shown inFIGS. 2A-2F. ⁵¹Cr release was determined in triplicate by γ-counting andthe percentage of specific lysis was calculated using the followingformula: [(experimental counts per minute (cpm) spontaneouscpm)/(maximal cpm spontaneous cpm)]×100. The results are shown in FIGS.2A-2F.

As shown in FIGS. 2A-2F, cells obtained from peripheral blood and sortedfor FD-1⁺ or TIM-3⁺ expression are capable of lysing the autologoustumor cell line.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A method of administering a cell populationenriched for tumor-reactive T cells to a mammal, the method comprising:(a) obtaining a bulk population of peripheral blood mononuclear cells(PBMCs) from a sample of peripheral blood; (b) specifically selectingCD8⁺ T cells that also express PD-1 and/or TIM-3 from the bulkpopulation; (c) separating the cells selected in (b) from unselectedcells to obtain a cell population enriched for tumor-reactive T cells;and (d) administering the cell population enriched for tumor-reactive Tcells to the mammal.
 2. The method of claim 1, wherein (b) furthercomprises specifically selecting CD8⁺ T cells that express PD-1 from thebulk population.
 3. A method of administering a cell population enrichedfor tumor-reactive T cells to a mammal, the method comprising: (a)obtaining a bulk population of peripheral blood mononuclear cells(PBMCs) from a sample of peripheral blood; (b) specifically selectingCD8⁺ T that are (i) TIM-3⁺/PD-1⁺, (ii) TIM-3⁻/PD-1⁺, or (iii)TIM-3⁺/PD-1⁻ from the bulk population; (c) separating the cells selectedin (b) from unselected cells to obtain a cell population enriched fortumor-reactive T cells; and (d) administering the cell populationenriched for tumor-reactive T cells to the mammal.
 4. The method ofclaim 1, wherein the cell population enriched for tumor-reactive T cellsis obtained without screening for autologous tumor recognition.
 5. Themethod of claim 1, wherein the bulk population of T cells is notnonspecifically stimulated prior to (b).
 6. The method of claim 1,further comprising expanding the numbers of T cells in the enriched cellpopulation obtained in (c).
 7. The method of claim 1, further comprisingculturing the enriched cell population obtained in (c) in the presenceof any one or more of TWS119, interleukin (IL-21), IL-12, IL-15, IL-7,transforming growth factor (TGF) beta, and AKT inhibitor (AKTi).
 8. Themethod of claim 1, further comprising stimulating the enriched cellpopulation obtained in (c) with a tumor antigen and/or with autologoustumor T cell.
 9. The method of claim 1, further comprising transducingor transfecting the cells of the enriched population obtained in (c)with a nucleotide sequence encoding any one or more of IL-12, IL-7,IL-15, IL-2, IL-21, mir155, and anti-PD-1 siRNA.
 10. A method oftreating cancer in a mammal, the method comprising administering a cellpopulation to the mammal by the method of claim 1 in an amount effectiveto treat cancer in the mammal.
 11. The method of claim 1, wherein (b)comprises specifically selecting CD8⁺ T cells that are TIM-3⁺/PD-1⁺ fromthe bulk population.
 12. The method of claim 1, wherein (b) comprisesspecifically selecting CD8⁺ T cells that are TIM-3⁺PD-1− from the bulkpopulation.
 13. The method of claim 3, wherein the cell populationenriched for tumor-reactive T cells is obtained without screening forautologous tumor recognition.
 14. The method of claim 3, wherein thebulk population of T cells is not nonspecifically stimulated prior to(b).
 15. The method of claim 3, further comprising expanding the numbersof T cells in the enriched cell population obtained in (c).
 16. Themethod of claim 3, further comprising culturing the enriched cellpopulation obtained in (c) in the presence of any one or more of TWS119,interleukin (IL-21), IL-12, IL-15, IL-7, transforming growth factor(TGF) beta, and AKT inhibitor (AKTi).
 17. The method of claim 3, furthercomprising stimulating the enriched cell population obtained in (c) witha tumor antigen and/or with autologous tumor T cell.
 18. The method ofclaim 3, further comprising transducing or transfecting the cells of theenriched population obtained in (c) with a nucleotide sequence encodingany one or more of IL-12, IL-7, IL-15, IL-2, IL-21, mir155, andanti-PD-1 siRNA.
 19. A method of treating cancer in a mammal, the methodcomprising administering a cell population to the mammal by the methodof claim 3 in an amount effective to treat cancer in the mammal.
 20. Amethod of treating cancer in a mammal, the method comprisingadministering a cell population to the mammal by the method of claim 2in an amount effective to treat cancer in the mammal.
 21. A method oftreating cancer in a mammal, the method comprising administering a cellpopulation to the mammal by the method of claim 4 in an amount effectiveto treat cancer in the mammal.
 22. A method of treating cancer in amammal, the method comprising administering a cell population to themammal by the method of claim 5 in an amount effective to treat cancerin the mammal.
 23. A method of treating cancer in a mammal, the methodcomprising administering a cell population to the mammal by the methodof claim 11 in an amount effective to treat cancer in the mammal.
 24. Amethod of treating cancer in a mammal, the method comprisingadministering a cell population to the mammal by the method of claim 12in an amount effective to treat cancer in the mammal.
 25. A method oftreating cancer in a mammal, the method comprising administering a cellpopulation to the mammal by the method of claim 13 in an amounteffective to treat cancer in the mammal.